We will investigate the stucture and distribution of two cytoskeletal polypeptides that are the first known intermediate filament-associated proteins of mammalian muscle. Intermediate filaments are a major component of eukaryotic cells. The cell type-specificity of intermediate filament protein subclasses makes them excellent molecular markers for tumor diagnosis. The function of intermediate filaments is unknown, partly due to lack of knowledge of intermediate filament associated proteins, which could cross-link filaments and modulate their physical properties. Preliminary studies demonstrate two cytoskeletal polypeptides with Mr of 200,000 and 220,000 and iso-electric points near pH 5.1 (HMW polypeptides), that are co-isolated with intermediate filaments from all bovine muscles. Purified HMW polypeptides bind directly to reconstituted desmin filaments with some specificity, since pilot studies demonstrate their co-precipitation with desmin but not actin filaments. Peptide mapping shows that the two HMW polypeptides are structurally related proteins, distinct from desmin, vimentin and the avian intermediate filament-associated protein, synemin. The first experiments examine some possible post-translational modifications of the HMW polypeptides, including phosphorylation and glycosylation. The second set of experiments will use HPLC-purified HMW polypeptides to examine their interactions with each other and their effects on intermediate filaments, including the stoichiometry of their saturating binding and the specificity of binding, as well as their effect on the critical concentration for desmin polymerization. The third project is preparation of polyclonal antibodies against the HMW polypeptides. The fourth set of experiments are immunochemical analyses of the cellular and tissue distribution of HMW polypeptides, to examine the nature of their association with the cytoskeleton in vivo, their extraction properties, and their cell-specificity and species-specificity. Human tumor samples will be stained with the anti-HMW antibodies, to test whether they differentially react with various neoplastic cells. (W)